The goal of this project is to elucidate the effect of pre-existing adeno-associated virus-2-specific T cell-mediated[unreadable] immunity on hepatic rAAV vector-mediated gene transfer. Recombinant (r) AAV-2 vectors have[unreadable] yielded promising results in gene replacement therapy of immunocornpetent experimental animals[unreadable] suggesting that they are comparatively poorly immunogenic. Humans are generally exposed to AAV-2[unreadable] antigens during childhood through infection by AAV-2 accompanying another virus, a so-called helper virus,[unreadable] such as an adenovirus or a herpes virus. Under circumstances where the poorly immunogenic AAV-2 is[unreadable] introduced with a helper virus that causes a strong inflammatory reaction, the helper virus is expected to[unreadable] provide an adjuvant effect that should facilitate the induction of a strong adaptive immune response to[unreadable] antigens of AAV-2. Human subjects unlike experimental rodents or canines are thus likely to have antigen-experienced[unreadable] T cells to AAV-2 that may become activated more readily upon rAAV-2-mediated gene transfer[unreadable] than has been appreciated by pre-clinical studies conducted thus far. Potentially confounding this problem is[unreadable] that AAV-2 persists in humans in a form that suggests that its genome may continue to be transcribed. Even[unreadable] very low levels of persistent AAV-2 antigen would maintain the immune system at a heightened stage of[unreadable] activation. Pre-existing T cell-mediated immunity to AAV-2 may also affect the use of simian AAVs for gene[unreadable] replacement therapy due to shared T cell epitopes between human and simian origin AAVs. One of the[unreadable] challenges of this application is to mimic natural AAV infection of humans in an experimental small animal[unreadable] species suited to conduct an in depth study of immune responses. For this, inbred mice will be used[unreadable] although they are not natural hosts for AAV-2 or its natural helper viruses. To overcome this limitation we[unreadable] developed and will continue to refine in aim 1 an experimental system to induce AAV capsid-specific T cell[unreadable] responses in mice, which resemble functionally those in human subjects. In aim 2, we will characterize the[unreadable] immune response to AAV-2 capsid in depth using the most suitable mouse model. In aim 3, we will use the[unreadable] model to test the effect of pre-existing immunity to AAV-2 on the efficacy, immunogenicity and longevity of[unreadable] rAAV-2-mediated gene transfer. Experiments will be extended in aim 4 to a vector system based on simianderived[unreadable] AAV-8 to assess if a heterologous rAAV vector can overcome pre-existing immunity to AAV-2.